靶向AML-LSC的新型嵌合抗原受体3A4-CAR基因的构建及优化

Leukemia stem cells (LSC) are the main cause of relapse due to their resistence to conventional chemotherapy. Recent studies on CD19 modified chimeric antigen receptor (CD19-CAR) T cells have demonstrated the promising results for refractory B-lineage malignancies. However, studies on the use of this technique based on CD45 isotype antibody for targeting AML and LSC have been NOT reported. Our previous findings showed that our new antibody ZCH (Zhejiang Children's Hospital)-6-3A4（3A4, muring IgG1），an anti-human CD45 isotype antibody, have the ability to recognize both newly diagnosed and relapsed leukemia cells. 3A4-NCTD (norcantharidin) immunotoxin studies have been proved to have the significant targeting killing effect on AML cells or therapeutic outcome of leukemia animal models. However, the affinity and the killing effect of this antibody will be significantly reduced after humanization. The aim of this project is to study the preparation of our novel monoclonal antibody based 3A4-CAR T cells consisting of 3A4-CD8-CD137 (4-1BB)-ζ，3A4-CD8-CD137-ζ-IL15，and suicide gene inducible caspase 9 (iCasp9) gene containing iCasp9-CD8-3A4-CD137-ζ and iCasp9-3A4-CD8-CD137-ζ-IL15 CAR genes which are to be inserted into the lentiviral vector pELNS to transfect the normal allogeneic peripheral blood mononuclear cells (MNC) to generate 3A4-CAR T cells. The targeting killing effects on AML cells and their LSCs by those 3A4-CAR modified T cells will be comprehensively studied and explored in both in vitro and in vivo settings using AML leukamia cell coculture and NOD/SCID mouse models for further optimization. It is expected that this study will provide the essential basis for 3A4-CARs to targeting the relapsed or refractory AML and their AML-LSCs, which will be of great important significance and clinical application potentials.

白血病干细胞（LSC）是该病复发的根源，常规化疗多耐药。CD19-嵌合抗原受体（CD19-CAR）T细胞在克服B系肿瘤耐药的临床试验中获成功，但用CD45亚类-CAR T细胞靶向AML及其LSC则国内外未见报道。研究发现，新单抗ZCH-6-3A4（简称3A4，CD45亚类）能识别初诊及复发难治AML及其LSC，有明显的靶向杀伤/治疗作用，但当该抗体经人源化改造后亲和力下降。本项目拟构建3A4-CD137-ζ CAR，插入慢病毒载体pELNS后转染正常人血MNC研制新型CAR-T细胞，通过体外实验和动物模型鉴定其对AML的靶向杀伤作用。并将iCaspase9自杀基因和/或IL-15基因整合到该CAR结构中，通过比较其扩增、杀伤能力及持续存活时间的变化，进一步优化并筛选功能最佳的3A4 CAR。本课题将为3A4-CAR T细胞治疗难治性AML奠定基础，具有十分重要的研究意义和潜在的应用前景。